Proteolysis by the 26S proteasome proceeds by binding ubiquitinated substrate protein to the 19S regulatory particle, followed by its deubiquitination, unfolding and translocation into the lumen of the 20S core, where it is degraded. In recent years, it has become evident that the proteasome is an appealing target for therapeutic intervention in cancer, immune-related disorders, inflammation, ischemic conditions, neurodegenerative disorders and other diseases. To date, the only FDA-approved proteasome inhibitor (VELCADE™) works by inhibiting the activity of the 20S core peptidases. Nonetheless, inhibiting other enzymatic activities residing within the proteasome complex could serve as equally effective, if not better, means of controlling proteasome function.
One such target candidate is the metalloprotease, Rpn11, residing within the 19S regulatory particle. It is responsible for the initial deubiquitination of target proteins (Eytan, et al. JBC 268(7):4668-4674 (1993); Verma, et al. Science 298:611-615 (2002)). The human homolog of this yeast protein is POH1. To date, a recombinant form of Rpn11 has not been generated. However, the deubiquitinating activity associated with Rpn11 can be distinguished in the context of a purified 26S proteasome complex by its sensitivity to ATP-γS and 1,10-phenanthroline (OPA) and insensitivity to 20S core protease inhibitors and the classic DUB inhibitors (ubiquitin aldehyde (UB-Al) or ubiquitin vinylsulphone (UbVS)).
In the pursuit to learn more about Rpn11 and its deubiquitinating activity, several assays have been employed. Most utilize purified 26S proteasome extracts as the source of enzyme and look for degradation in the presence or absence of a DUB inhibitor. Detecting Rpn11 activity required a ubiquitinated protein such as Ub-sic 1 (Verma et al., supra) and the reaction was monitored by SDS-PAGE followed by immunoblotting with either α-ubiquitin (α-Ub) or α-protein such as α-sic 1. Substrates that were radio- or fluorescent-labeled still required a step that separated uncleaved substrate from cleaved product such as PAGE (Eytan et al., supra) or TCA-precipitation (Yao et al., Nature 419: 403-407 (2002)), respectively.
Thus, a high throughput (HTP) assay employing novel Rpn11 substrate proteins is highly desirable in order to screen large numbers of molecules to identify modulators of Rpn11 activity.